Immunoreactive peptides from Epstein-Barr virus

ABSTRACT

The Epstein-Barr virus (EBV) specific polypeptides ETFTETWNRFITHTE (SEQ. ID NO: 1), GMLEASEGLDGWIHQ (SEQ. ID NO:2), HQQGGWSTLIEDNIP (SEQ. ID NO:3), and KQKHPKKVKQAFNPL (SEQ. ID NO:4) are among those disclosed. Also disclosed are the use of these polypeptides for the production of polypeptide-specific antibodies and the diagnosis and treatment of EBV-associated disease.

CROSS-REFERENCE TO RELATED APPLICATION

This patent application is a divisional of U.S. application Ser. No.10/442,456 filed May 21, 2003, now U.S. Pat. No. 7,326,535, issued Feb.5, 2008, which is a divisional of U.S. application Ser. No. 08/392,934,filed on Oct. 28, 1996, now U.S. Pat. No. 7,060,283, issued Jun. 13,2006, which is the National Phase application of International Patentapplication Serial Number PCT/US93/08699, filed on Sep. 15, 1993, whichclaims priority of U.S application Ser. No. 07/945,280, filed Sep. 15,1992 (abandoned). The disclosures of each of the foregoing applicationsare hereby incorporated by reference in their entirety.

Subject matter described herein was made with government support undergrant No. CA039617 awarded by the National Institutes of Health. TheUnited States government may have certain rights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to the diagnosis and therapy of Epstein-Barrvirus associated disease. More specifically, these modalities arefounded on the discovery of EBV-specific peptides.

2. Description of Related Art

Epstein-Barr virus (EBV) is a human herpesvirus which is endemic in allhuman populations. Most people are infected with the virus in earlychildhood and then carry the virus for life. If the initial infection isdelayed until adolescence, infectious mononucleosis (IM) frequentlyresults. The virus is also linked with certain kinds of cancer. In themalarial belt of Africa, EBV is a contributory factor in the developmentof Burkitt's lymphoma and in South-East Asia, the virus is linked to thehigh incidence of undifferentiated nasopharyngeal carcinomas.

Acute viral infection leads to the production of specific nuclearantigens (termed EBNA-I and EBNA-II), an “early antigen” (EA) complex,viral capsid antigens (VCA), and other associated molecules. The “earlyantigen complex” consists of the “early antigen-diffuse” (EA-D) and the“early antigen-restricted” (EA-R) antigens, based on their distributionin immunofluorescence assays in the cytoplasm plus nucleus (i.e. diffusedistribution) or in the cytoplasm only (i.e. restricted) and based ontheir staining appearance in methanol-fixed cells. These EA antigens,with molecular weight 50-55 Kd, 17 Kd, and 85 Kd, respectively, aresynthesized during the “lytic” phase of EBV infection and not intransformed lymphoblastoid cells. Antibodies against the early antigensare present during acute EBV infection and then disappear as the virusenters a phase of latency. The reappearance of anti-EA antibodiessignals viral reactivation and provides a clue to the possible role ofthis virus in diseases such as nasopharyngeal carcinoma and Burkitt'slymphoma.

Indirect evidence has suggested a possible role for EBV reactivation inpatients with Sjogren's syndrome, an autoimmune disorder characterizedby lymphoid infiltrates of the salivary gland (the normal site for EBVlatency). Since antibodies to EA antigens are detected byimmunofluorescence assays, such antibodies cannot be detected inpatients who possess antinuclear and anticytoplasmic antibodies as partof an autoimmune disease. Therefore it would be desirable to havepurified EA molecules to allow measurement of anti-EA antibodies inpatients with autoimmune diseases and to more accurately quantitateanti-EA antibodies in other patients with acute or reactivated EBV.

Recently, the DNA sequence of EBV was determined (Beer, et al., Nature310:207, 1984) and the EA-D antigen localized in the genome. Using amonoclonal antibody directed against the EA-D protein, sufficientprotein was purified to allow partial amino acid sequence determinationand thus localization of the coding sequences. Using that information,it was possible to prepare a series of synthetic peptides based on theDNA sequence. The same strategy has proved useful in identifyingimmunologically important epitopes on the EBNA-I antigen (Rhodes, etal., J. Immunol., 134:211, 1985) and the EBNA-II antigens (Dinner, J.Proc. Natl. Acad. Sci. U.S.A. 81:4652, 1984) of EBV. A synthetic peptidederived from the EA-D molecule which contains an epitope reactive withimmune human sera from patients with IM and other disease states hasalso been described (Fox, et al., J. Clin. Lab. Anal., 1: 140, 1987).

Recent studies have shown that chemically synthesized polypeptidescorresponding to short linear segments of a protein's primary amino acidresidue sequence can be used to induce antibodies that immunoreact withthe native protein (Lerner, et al., Nature, 299:592, 1982; Sutcliffe, etal., Science, 219:260, 1983). In addition, some studies have shown thatsynthetic polypeptides can immunoreact with antibodies induced by nativeproteins (Rhodes, et al., J. Immunol., 134:211, 1985). Thus, somesynthetic polypeptides can immunologically mimic the immunogenic andantigenic determinants of native proteins.

However, as is well known in the art, the application of syntheticpeptide technology still suffers several shortcomings. For instance, theidentification of peptides capable of mimicking antigenic determinantson a native protein requires knowing the amino acid residue sequence ofthe protein. Whereas the amino acid residue sequence can be predictedfrom the nucleic acid sequence of the gene coding for the protein, sucha prediction can only be made if the correct reading frame of the geneis known.

The nucleic acid sequence of the EBV genome is known. However, even if aprotein's amino acid residue sequence is known, methods for identifyingthe loci in the protein that constitute the immunogenic and antigenicdeterminants are experimental in nature and do not yield predictableresults. There are at least two reasons for this. First, without knowinga protein's 3-D structure there is no reliable method for determiningwhich linear segments of the protein are accessible to the host's immunesystem. Second, whether the 3-D structure is known or not, short linearpolypeptides often appear not to have the ability to mimic the requiredsecondary and tertiary conformational structures to constituteappropriate immunogenic and antigenic determinants (Tamer, et al.,Nature, 312:127, 1984). However, methods such as Berzofsky's algorithm(AMPHI program, 1987) have been developed which allow the identificationof those dominant epitopes of a molecule that preferentially interactwith T or B-cells.

Previous studies have examined the cellular immune response toEBV-induced antigens synthesized during the virus replication cycle(Pothen, et al., Int. J. Cancer, 49:656, 1991). The results demonstratedthat some of the components of the early antigen (EA) complex were veryeffective in inducing a strong T-cell proliferative response similar tothat previously noted with the major membrane glycoprotein, gp350/250(Ulaeto, et al., Europ. J. Immunol., 18:1689, 1988). Both CD4+ and CD8+lymphocyte populations from EBV-infected donors proliferated in thepresence of polypeptides purified from the EA complex by immunoaffinitychromatography. The major polypeptide of EA-D and one of the majorpolypeptides of EA-R were particularly effective in this T-cellrecognition assay. The data suggested that these components of the EAcomplex might function as important target antigens in theimmunosurveillance of EBV-infected or immortalized cells. Identificationof the dominant T and B-cell epitopes expressed on EA-R complexpolypeptides would provide information on the importance of the antibodyresponses to these components in the diagnosis and management ofindividuals with EBV-associated lymphoproliferative diseases.

It would be desirable to develop improved methods to assay for thepresence of EA-R or EA-D and anti-EA-R or anti-EA-D antibodies in a bodysample so as to allow diagnosis of EBV involvement in disease, as wellas diagnosis os the stage of a disease such as infectious mononucleosis.The identification of those B and T-cell epitopes on EA-R/EA-Dpolypeptides would be an important step toward synthesis of themolecules for use for diagnostic and disease management purposes inindividuals with EBV associated lymphoproliferative diseases.

SUMMARY OF THE INVENTION

The present invention provides polypeptides which define immunogenicsites which are specific for Epstein-Barr virus (EBV). Thesepolypeptides can be used for immunodiagnosis and immunotherapy ofEBV-associated diseases and to produce monoclonal antibodies whichspecifically bind to these polypeptides. These monoclonal antibodies canbe used to detect antigen comprising the polypeptide of the inventionand also used therapeutically to ameliorate EBV-associated disease.

The details of the preferred embodiment of the present invention are setforth in the accompanying drawings and tire description below. Once thedetails of the invention are known, numerous additional innovations andchanges will become obvious to one skilled in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic map of the EBV genome denoting the BamHI HRF1reading frame and the regions in BHRF1 encoding for the syntheticpeptides.

FIGS. 2A, 2B, and 2C shows the proliferation response of PBL from threeanti-VCA-positive, anti-EA-negative individuals (A-C) to differentconcentrations of the three p17 synthetic peptides.

FIGS. 3A and 3B shows proliferation response of CD4+ and CD8+ T-cellsubpopulations from two donors (A and B) to different concentrations ofthe synthetic peptide, P17.1.

FIG. 4 shows serological reactivity of different sera with the p17synthetic peptides (17.1, 17.2, 17.3). All sera were tested in the ELISAat 1:10 dilution. Numbers in parenthesis in each column represent numberof unreactive sera (absorbance<0.1).

FIG. 5 shows serological reactivity of anti-EA positive sera fromdifferent disease categories against the p17 synthetic peptides. Allsera were tested at 1:10 dilution. ABL African Burkitt's lymphoma;NANHL, North American non-Hodgkin's lymphomas including intermediategrade large cell lymphomas, or high grade B cell lymphoma. NANPC, NorthAmerican nasopharyngeal carcinoma. Numbers in parenthesis in each columnrepresent number of unreactive sera (absorbance<0.1).

DETAILED DESCRIPTION OF THE INVENTION

A preferred embodiment of the invention comprises the eptiopicpolypeptides ETFTETWNRFITHTE (SEQ. I.D. NO. 1), GMLEASEGLDGWIHQ (SEQI.D. NO. 2), HQQGGWSTLIEDNIP (SEQ. I.D. NO. 3), KQKHPKKVKQAFNPL (SEQ.I.D. NO. 4), and conservative variations and mixtures of these peptides.The term “conservative variation” as used herein denotes the replacementof an amino acid residue by another, biologically similar residue.Examples of conservative variations include the substitution of onehydrophobic residue such as isoleucine, valine, leucine or methioninefor another, or the substitution of one polar residue for another, suchas the substitution of arginine for lysine, glutamic for aspartic acids,or glutamine for asparagine, and the like. The term “conservativevariation” also includes the use of a substituted amino acid in place ofan unsubstituted parent amino acid provided that antibodies raised tothe substituted polypeptide also immunoreact with the unsubstitutedpolypeptide. Thus, by using a routine screening method, such as bytesting a conservative variant polypeptide with sera from a patient withEBV-associated disease, one of skill in the art can readily determine ifthe variant polypeptide has the requisite biological activity of thepolypeptide of the invention without resort to undue experimentation.

The epitopic polypeptides of the invention may contain additional aminoacids at the amino and carboxy termini in order to increase theirserological reactivity. Preferably, the additional amino acids are thenaturally occurring amino acids of the protein, on conservativevariations of these amino acids, and range in number from about 0 toabout 5 independently. For example, a variation of SEQ. I.D. NO. 1comprises the epitopic polypeptide, QNSETFTETWNRFITHTEHVD (SEQ. I.D. NO.5), and a variation of SEQ. I.D. NO. 4 comprises the epitopicpolypeptide, ARQKQKHPKKVKQAFNPLI (SEQ. I.D. NO. 6), where the underlinedamino acids represent extensions of the original polypeptides. Thepolypeptides of the invention can also be utilized as repeating unitsranging from 1 to about 1000 units in length. These units can behomogeneous, for example, where all of the units are repeats of the samepolypeptide or can be mixtures of the polypeptides of the invention.

The peptides of the invention can be used singularly, in mixtures, or asmultimers such as aggregates, polymers, and the like. Thus, theinvention embraces polypeptides which comprise one or more of the same,or different, polypeptides of the invention to produce a homogeneous orheterogeneous polymer with respect to the particular polypeptides of theinvention which are contained therein. Appropriate techniques forproducing various mixtures, aggregates, multimers and the like will beknown to those of skill in the art. For example, the invention includesa polypeptide comprising SEQ. I.D. NO.1 and SEQ. I.D. NO.2, NO.3 or NO.4or any combination of these, wherein the sequences are linked directlyor indirectly, for example, by using a spacer or linker moiety.

Peptides of the invention can be synthesized by such well known solidphase peptide synthesis methods described by Merrifield, J. Am. Chem.Soc. 85:2149, 1962, and Stewart and Young, Solid Phase PeptidesSynthesis, (Freeman, San Francisco, 1969, pp. 27-62), using acopoly(styrene-divinylbenzene) containing 0.1-1.0 mMol amines/g polymer.On completion of chemical synthesis, the peptides can be deprotected andcleaved from the polymer by treatment with liquid HF-10% anisole forabout ¼-1 hours at 0° C. After evaporation of the reagents, the peptidesare extracted from the polymer with 1% acetic acid solution which isthen lyophilized to yield the crude material. This can normally bepurified by such techniques as gel filtration on SEPHADEX G-15 using 5%acetic acid as a solvent. Lyophilization of appropriate fractions of thecolumn will yield the homogeneous peptide or peptide derivatives, whichcan then be characterized by such standard techniques as amino acidanalysis, thin layer chromatography, high performance liquidchromatography, ultraviolet absorption spectroscopy, molar rotation,solubility, and quantitated by the solid-phase Edman degradation.

During or after the synthesis, reactive amino acids may be protected byvarious blocking groups, for example, cysteines may be blocked by3,4-dimethylbenzyl (DMB) groups, arginines and histidines by tosyl (TOS)groups, aspartic acid and glutamic acids by benzyl (Bzl) groups, andlysines the 2-chloro-benzyloxycarboxyl (2-CBZ) groups. Other protectiveblocking groups are well-known, and can be used in the presentinvention. Those of ordinary skill in the art will know of othertechniques for peptide synthesis, or can readily ascertain suchtechniques, without resorting to undue experimentation.

Alternatively, the polypeptides of the invention can be produced usingrecombinant techniques commonly known to those of skill in the art (see,for example, Current Protocols in Molecular Biology, Ausubel, et al.,eds., Wiley Interscience Press, 1989, incorporated herein by reference).

The invention also provides polynucleotides which encode thepolypeptides of the invention. As used herein, “polynucleotide” refersto a polymer of deoxyribonucleotides or ribonucleotides, in the form ofa separate fragment or as a component of a larger construct. DNAencoding a peptide of the invention can be assembled from cDNA fragmentsor from oligonucleotides which provide a synthetic gene which is capableof being expressed in a recombinant transcriptional unit. Polynucleotidesequences of the invention include DNA, RNA and cDNA sequences. Apolynucleotide sequence can be deduced from the genetic code, however,the degeneracy of the code must be taken into account. Polynucleotidesof the invention include sequences which are degenerate as a result ofthe genetic code. The invention also includes sequences which arecomplementary and can hybridize to the polynucleotides which encode thepolypeptides of the invention.

The term “EBV-associated disease” means any disease caused, directly orindirectly, by EBV as well as diseases which predispose a patient toinfection by EBV. Examples of diseases falling into the former categoryinclude infectious mononucleosis, nasopharyngeal carcinoma, andBurkitt's lymphoma. Diseases in the latter category (i.e., those whichplace the patient at risk of EBV infection) include Sjorgren's syndromeand, generally, any condition that causes a state of immunosuppressionor decreased function of the immune system such as patients who receiveorgan transplants and certain cancer therapies.

The present invention further relates to monoclonal antibodies which arespecific for the polypeptides of the invention as well as the diagnosticand therapeutic use of these monoclonal antibodies. This specificityenables the monoclonal antibody, and like monoclonal antibodies withlike specificity, to be used to bind the polypeptide of the inventionwhen the polypeptide, or amino acids comprising the polypeptide, arepresent in specimens or a host, such as a human.

Numerous techniques can be utilized to produce the monoclonal antibodiesof the invention without resorting to undue experimentation. To a greatextent, the products of such monoclonal antibodies is rendered routinebecause of the highly defined nature of the polypeptides of theinvention. Thus, whether the polypeptides of the invention are used forimmunization and/or screening, the very limited number of immunogenicdeterminants on the polypeptides greatly simplifies the identificationof cell lines producing monoclonal antibodies of the invention, forexample, by limiting the repertoire of clonal expression possible.

One very useful type of cell line for expression of the monoclonalantibodies of the invention is the hybridoma. The general method usedfor production of hybridomas producing monoclonal antibody is well known(Kohler and Milstein, Nature, 256:495, 1975). The resulting hybridomaswere then screened for production of monoclonal antibodies capable ofbinding to the polypeptides of the invention.

The techniques of sensitization and/or immunization, cell fusion,ascites production, selection of mixed hybridomas, or subcloning ofmonoclonal hybridomas are generally well known in the art. Attention isdirected to 1.0 Koprowski, et al., U.S. Pat. No. 4,172,124, Koprowski,et al., U.S. Pat. No. 4,196,265, or Douillard, J. Y. and Hoffman, T.,Basic Facts about Hybridomas, in Compendium of Immunology, Vol. II, L.Schwartz, ed. (1981), which are herein incorporated by reference.

In general, the purified epitopic peptides have a cystine attached atthe C-terminus to permit unidirectional attachment of the syntheticpeptide to an immunogenic protein through a connecting bridge, forexample, maleimidobenzoylated (MB)-keyhole limpet hemocyanin (KLH).Other immunogenic conjugates can also be used, for example, albumin, andthe like. The resulting structure may have several peptide structureslinked to one molecule of protein.

Somatic cells derived from a host immunized against the syntheticpeptides can be obtained by any suitable immunization technique. Thehost subject is immunized by administering the antigen, usually in theform of a protein conjugate, as indicated above, by any suitable method,preferably by injection, either intraperitoneally, intravenously,subcutaneously, or by intra-foot pad. Adjuvants may be included in theimmunization protocol.

The initial immunization with the protein bound antigen can be followedby several booster injections given periodically at intervals of severalweeks. The antibody contained in the plasma of each host can then betested for its reactivity with the immunizing polypeptide of theinvention. The host having the highest response is usually mostdesirable as the donor of the antibody secreting somatic cells used inthe production of hybridomas. Alternatively, hyperimmunization can beeffected by repeatedly injecting additional amounts of peptide-proteinconjugate by intravenous and/or intraperitoneal route.

The isolation of hybridomas producing monoclonal antibodies of theinvention can be accomplished using routine screening techniques whichpermit determination of the elementary reaction pattern of themonoclonal antibody of interest. Thus, if a monoclonal antibody beingtested binds with a polypeptide of the invention, then the antibodybeing tested and the antibody produced by the hybridomas of theinvention are equivalent.

Alternatively, since the invention teaches polypeptides or amino acidsequences which are specifically required for binding of the preferredmonoclonal antibodies of the invention, it is now possible to use thesepeptides for purposes of immunization to produce hybridomas which, inturn, produce monoclonal antibodies specific for the polypeptide. Thisapproach has the added advantage of decreasing the repertoire ofmonoclonal antibodies generated by limiting the number of antigenicdeterminants presented at immunization by the polypeptide. Themonoclonal antibodies produced by this method can be screened forspecificity using standard techniques, for example, by bindingpolypeptide to a microtiter plate and measuring binding of themonoclonal antibody by an ELISA assay.

It is also possible to determine, without undue experimentation, if amonoclonal antibody has the same specificity as a monoclonal antibody ofthe invention by ascertaining whether the former prevents the latterfrom binding the polypeptide of the invention. If the monoclonalantibody being tested competes with the monoclonal antibody of theinvention, as shown by a decrease in binding by the monoclonal antibodyof the invention, then it is likely that the two monoclonal antibodiesbind to the same, or a closely related, epitope.

Still another way to determine whether a monoclonal antibody has thespecificity of a monoclonal antibody of the invention is to pre-incubatethe monoclonal antibody of the invention with the polypeptide of theinvention with which it is normally reactive, and then add themonoclonal antibody being tested to determine if the monoclonal antibodybeing tested is inhibited in its ability to bind the antigen. If themonoclonal antibody being tested is inhibited then, in all likelihood,it has the same, or a closely related, epitopic specificity as themonoclonal antibody of the invention.

While the in vivo use of a monoclonal antibody from a foreign donorspecies in a different host recipient species is usually uncomplicated,a potential problem which may arise is the appearance of an adverseimmunological response by the host to antigenic determinants present onthe donor antibody. In some instances, this adverse response can be sosevere as to curtail the in vivo use of the donor antibody in the host.Further, the adverse host response may serve to hinder theEBV-associated disease suppressing efficacy of the donor antibody. Oneway in which it is possible to circumvent the likelihood of an adverseimmune response occurring in the host is by using chimeric antibodies(Sun, et al., Hybridoma, 5 (Supplement 1): S17, 1986; Oi, et al., BioTechniques, 4(3): 214, 1986). Chimeric antibodies are antibodies inwhich the various domains of the antibodies' heavy and light chains arecoded for by DNA from more than one species. Typically, a chimericantibody will comprise the variable domains of the heavy (V_(H)) andlight (V_(L)) chains derived from the donor species producing theantibody of desired antigenic specificity, and the variable domains ofthe heavy (C_(H)) and light (C_(L)) chains derived from the hostrecipient species. It is believed that by reducing the exposure of thehost immune system to the antigenic determinants of the donor antibodydomains, especially those in the C_(H) region, the possibility of anadverse immunological response occurring in the recipient species willbe reduced. Thus, for example, it is possible to produce a chimericantibody for in vivo clinical use in humans which comprises mouse V_(H)and V_(L) domains coded for by DNA isolated from a hybridoma of theinvention and C_(H) and C_(L) domains coded for with DNA isolated from ahuman leukocyte.

Under certain circumstances, monoclonal antibodies of one isotype mightbe more preferable than those of another in terms of their diagnostic ortherapeutic efficacy. For example, from studies on antibody-medicatedcytolysis, it is known that unmodified mouse monoclonal antibodies ofisotype gamma-2a and gamma-3 are generally more effective in lysingtarget cells than are antibodies of the gamma-1 isotype. Thisdifferential efficacy is thought to be due to the ability of thegamma-2a and gamma-3 isotypes to more actively participate in thecytolytic destruction of target cells. Particular isotypes of amonoclonal antibody can be prepared either directly, by selecting fromthe initial fusion, or prepared secondarily, from a parental hybridomasecreting a monoclonal antibody of different isotype by using the sibselection technique to isolate class-switch variants (Steplewski, etal., Proc. Natl. Acad. Sci., U.S.A., 82:8653, 1985; Spira, et al., J.Immunol. Methods, 74:307, 1984). Thus, the monoclonal antibodies of theinvention would include class-switch variants having specificity for apolypeptide of the invention.

The isolation of other hybridomas secreting monoclonal antibodies withthe specificity of the monoclonal antibodies of the invention can alsobe accomplished by one of ordinary skill in the art by producinganti-idiotypic antibodies (Herlyn, et al., Science, 232:100, 1986). Ananti-idiotypic antibody is an antibody which recognizes uniquedeterminants present on the monoclonal antibody produced by thehybridoma of interest. These determinants are located in thehypervariable region of the antibody. It is this region which binds to agiven epitope and, thus, is responsible for the specificity of theantibody. An anti-idiotypic antibody can be prepared by immunizing ananimal with the monoclonal antibody of interest. The immunized animalwill recognize and respond to the idiotypic determinants of theimmunizing antibody and produce an antibody to these idiotypicdeterminants. By using the anti-idiotypic antibodies of the immunizedanimal, which are specific for the monoclonal antibody produced by asingle hybridoma which was used to immunize the second animal, it is nowpossible to identify other clones with the same idiotype as the antibodyof the hybridoma used for immunization.

Idiotypic identity between monoclonal antibodies of two hybridomasdemonstrates that the two monoclonal antibodies are the same withrespect to their recognition of the same epitopic determinant. Thus, byusing anti-idiotypic antibodies, it is possible to identify otherhybridomas expressing monoclonal antibodies having the same epitopicspecificity.

It is also possible to use the anti-idiotype technology to producemonoclonal antibodies which mimic an epitope. For example, ananti-idiotypic monoclonal antibody made to a first monoclonal antibodywill have a binding domain in the hypervariable region which is the“image” of the epitope bound by the first monoclonal antibody. Thus, theanti-idiotypic monoclonal antibody could be used for immunization sincethe anti-idiotype monoclonal antibody binding domain effectively acts asan antigen.

Especially preferred for in vivo diagnosis and therapy of EBV-associateddisease are human monoclonal antibodies. Recent advances in themonoclonal antibody art now make possible the ability to readily producehuman monoclonal antibodies by using recombinant cloning techniques.Typically, these techniques utilize lymphocytes from a patient withdemonstrated antibody to the antigen of interest followed by thegeneration of a recombinatorial library from the nucleic acid isolatedfrom these lymphocytes. This library contains an expression vectoradapted to allow the cloning of immunoglobulin heavy and light chains inthe host organism. Individual colonies producing an antibody of desiredspecificity for a particular antigen are identified, for example, byattaching the antigens to a solid place and “panning” for the antibody.(See, for example, Burton, et al., Proc. Natl. Acad. Sci. USA, 88:10134,1991, which is incorporated herein by reference).

Thus, one of skill in the art can analogously produce human monoclonalantibodies specific for the peptides of the invention as a matter ofroutine by generating a recombinatorial library using nucleic acid,preferably mRNA, from the lymphocytes of an individual with humoralimmunity to EBV and screening the library so produced using thepolypeptides of the invention. In light of the highly defined nature ofthe polypeptides of the invention, each polypeptide will possess few(probably 1 or 2) epitopes and the screening of the library can be donein a simple and highly specific manner without requiring undueexperimentation.

The term “antibody” as used in this invention is meant to include intactmolecules as well as fragments thereof, such as Fab and F(ab′)₂, whichare capable of binding the epitopic determinant.

The monoclonal antibodies of the invention can be used in any animal inwhich it is desirable to administer in vitro or in vivo immunodiagnosisor immunotherapy. The term “animal” as used herein is meant to includeboth humans as well as non-humans.

The monoclonal antibodies of the invention are suited for use, forexample, in immunoassays in which they can be utilized in liquid phaseor bound to a solid phase carrier. In addition, the monoclonalantibodies in these immunoassays can be detectably labeled in variousways. Examples of types of immunoassays which can utilize monoclonalantibodies of the invention are competitive and non-competitiveimmunoassays in either a direct or indirect format. Examples of suchimmunoassays are the radioimmunoassay (RIA) and the sandwich(immunometric) assay. Detection of the antigens using the monoclonalantibodies of the invention can be done utilizing immunoassays which arerun in either the forward, reverse, or simultaneous modes, includingimmunohistochemical assays on physiological samples. Those of skill inthe art will know, or can readily discern, other immunoassay formatswithout undue experimentation.

The monoclonal antibodies of the invention can be bound to manydifferent carriers and used to detect the presence of an antigencomprising a polypeptide of the invention. Examples of well-knowncarriers include glass, polystyrene, polypropylene, polyethylene,dextran, nylon, amylases, natural and modified celluloses,polyacrylamides, agaroses and magnetite. The nature of the carrier canbe either soluble or insoluble for purposes of the invention. Thoseskilled in the art will know of other suitable carriers for bindingmonoclonal antibodies, or will be able to ascertain such, using routineexperimentation.

There are many different labels and methods of labeling known to thoseof ordinary skill in the art. Examples of the types of labels which canbe used in the present invention include enzymes, radioisotopes,fluorescent compounds, colloidal metals, chemiluminescent compounds,phosphorescent compounds, and bioluminescent compounds. Those ofordinary skill in the art will know of other suitable labels for bindingto the monoclonal antibody, or will be able to ascertain such, usingroutine experimentation. Furthermore, the binding of these labels to themonoclonal antibody of the invention can be done using standardtechniques common to those of ordinary skill in the art.

For purposes of the invention, an antibody specific for a polypeptide ofthe invention or an antigen comprising a polypeptide of the inventionmay be detected by the monoclonal antibodies of the invention whenpresent in biological fluids and tissues. Any specimen containing adetectable amount of such antigen can be used. A sample can be a liquidsuch as urine, saliva, cerebrospinal fluid, blood, serum and the like,or a solid or semi-solid such as tissues, feces, and the like, or,alternatively, a solid tissue such as those commonly used inhistological diagnosis. An especially preferred sample is blood.

Another technique which may also result in greater sensitivity consistsof coupling the antibodies to low molecular weight haptens. Thesehaptens can then be specifically detected by means of a second reaction.For example, it is common to use such haptens as biotin, which reactswith avidin, or dinitrophenyl, pyridoxal, and fluorescein, which canreact with specific anti-hapten antibodies.

As used in this invention, the term “epitope” is meant to include anydeterminant capable of specific interaction with the monoclonalantibodies of the invention. Epitopic determinants usually consist ofchemically active surface groupings of molecules such as amino acids orsugar side chains and usually have specific three dimensional structuralcharacteristics, as well as specific charge characteristics.

In using the monoclonal antibodies of the invention for the in vivodetection of antigen, the detectably labeled monoclonal antibody isgiven in a dose which is diagnostically effective. The term“diagnostically effective” means that the amount of detectably labeledmonoclonal antibody is administered in sufficient quantity to enabledetection of the site having the antigen comprising a polypeptide of theinvention for which the monoclonal antibodies are specific.

The concentration of detectably labeled monoclonal antibody which isadministered should be sufficient such that the binding to those cellshaving the polypeptide is detectable compared to the background.Further, it is desirable that the detectably labeled monoclonal antibodybe rapidly cleared from the circulatory system in order to give the besttarget-to-background signal ratio.

As a rule, the dosage of detectably labeled monoclonal antibody for invivo diagnosis will vary depending on such factors as age, sex, andextent of disease of the individual. The dosage of monoclonal antibodycan vary from about 0.01 mg/m² to about 500 mg/m², preferably 0.1 mg/m²to about 200 mg/m², most preferably about 0.1 mg/m² to about 10 mg/m².Such dosages may vary, for example, depending on whether multipleinjections are given, antigenic burden, and other factors known to thoseof skill in the art.

For in vivo diagnostic imaging, the type of detection instrumentavailable is a major factor in selecting a given radioisotope. Theradioisotope chosen must have a type of decay which is detectable for agiven type of instrument. Still another important factor in selecting aradioisotope for in vivo diagnosis is that the half-life of theradioisotope be long enough so that it is still detectable at the timeof maximum uptake by the target, but short enough so that deleteriousradiation with respect to the host is minimized. Ideally, a radioisotopeused for in vivo imaging will lack a particle emission, but produce alarge number of photons in the 140-250 keV range, which may be readilydetected by conventional gamma cameras.

For in vivo diagnosis radioisotopes may be bound to immunoglobulineither directly or indirectly by using an intermediate functional group.Intermediate functional groups which often are used to bindradioisotopes which exist as metallic ions to immunoglobulins are thebifunctional chelating agents such as diethylenetriaminepentacetic acid(DTPA) and ethylenediaminetetraacetic acid (EDTA) and similar molecules.Typical examples of metallic ions which can be bound to the monoclonalantibodies of the invention are ¹¹¹In, ⁹⁷Ru, ⁶⁷Ga, ⁶⁸Ga, ⁷²As, ⁸⁹Zr, and²⁰¹Tl.

The monoclonal antibodies of the invention can also be labeled with aparamagnetic isotope for purposes of in vivo diagnosis, as in magneticresonance imaging (MRI) or electron spin resonance (ESR). In general,any conventional method for visualizing diagnostic imaging can beutilized. Usually gamma and positron emitting radioisotopes are used forcamera imaging and paramagnetic isotopes for MRI. Elements which areparticularly useful in such techniques include ¹⁵⁷Gd, ⁵⁵Mn, ¹⁶²Dy, ⁵²Cr,and ⁵⁶Fe.

The monoclonal antibodies of the invention can be used in vitro and invivo to monitor the course of amelioration of EBV-associated disease inan animal. Thus, for example, by measuring the increase or decrease inthe number of cells expressing antigen comprising a polypeptide of theinvention or changes in the concentration of such antigen presentvarious body fluids, it would be possible to determine whether aparticular therapeutic regimen aimed at ameliorating the EBV-associateddisease is effective.

The term “ameliorate” denotes a lessening of the detrimental affect ofthe EBV-associated disease in the animal receiving therapy. The term“therapeutically effective” means that the amount of monoclonal antibodyor polypeptide used is of sufficient quantity to ameliorate theEBV-associated disease.

The term “immunogenically effective amount,” as used in the invention,that amount of polypeptide which is necessary to induce an ameliorativeimmune response to the EBV-associated disease, for example, bystimulating the production of antibodies which will bind to an antigencomprising a polypeptide of the invention.

In inducing an immune response to a polypeptide of the invention, thepolypeptide can be administered parenterally by injection, rapidinfusion, nasopharyngeal absorption, dermal absorption, and orally.Preparations for parenteral administration include sterile or aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Carriers for occlusive dressings can be used to increaseskin permeability and enhance antigen absorption. Liquid dosage formsfor oral administration may generally comprise a liposome solutioncontaining the liquid dosage form. Suitable forms for suspending theliposomes include emulsions, suspensions, solutions, syrups, and elixirscontaining inert diluents commonly used in the art, such as purifiedwater. Besides the inert diluents, such compositions can also includeadjuvants, wetting agents, emulsifying and suspending agents, andsweetening, flavoring, and perfuming agents.

It is also possible for the antigenic preparations containingpolypeptides of the invention to include an adjuvant. Adjuvants aresubstances that can be used to non-specifically augment a specificimmune response. Normally, the adjuvant and the antigen are mixed priorto presentation to the immune system, or presented separately, but intothe same site of the animal being immunized. Adjuvants can be looselydivided into several groups based on their composition. These groupsinclude oil adjuvants (for example, Freund's Complete and Incomplete),mineral salts (for example, AlK(SO₄)₂, AlNa(SO₄)₂, AlNH₄(SO₄), silica,alum, Al(OH)₃, Ca₃(PO₄)₂, kaolin, and carbon), polynucleotides (forexample, poly IC and poly AU acids), and certain natural substances (forexample, wax D from Mycobacterium tuberculosis, as well as substancesfound in Corynebacterium parvum, Bordetella pertussis, and members ofthe genus Brucelia).

The physical form of the polypeptide antigen which is used to immunizean animal can be either aggregated or non-aggregated. Aggregatedpolypeptide can be produced from non-aggregated polypeptide by suchcommon techniques as, for example, treatment with glutaraldehyde orother cross-linking agents. The aggregated polypeptide thus derivedcould then be used for purposes of producing an EBV-associated diseaseameliorating composition effective in inducing an active immunereaction. However, regardless of whether an animal is immunized withaggregated or non-aggregated, both of these forms of polypeptide shouldcause the production of antibodies to the polypeptide. Thus, it ispossible to use these anti-polypeptide antibodies diagnostically as, forexample, in a kit to detect the presence of polypeptide in a specimen.

As described above, the polypeptide antigen preparations of theinvention can be used to induce the production of antibodies which willbind to epitopic determinants of the polypeptide. A particularly usefulmethod in enhancing the production of antibodies to polypeptide is tofirst immunize with the polypeptide antigenic preparation of theinvention followed by a later immunization. Many different techniquesexist for the timing of the immunizations when a multiple immunizationregimen is utilized. It is possible to use the antigenic preparation ofthe invention more than once to increase the levels and diversity ofexpression of the immunoglobulin repertoire expressed by the immunizedanimal. Typically, if multiple immunizations are given, they will bespaced one to two months apart. Generally, the dosage of polypeptideadministered to an animal will vary depending on such factors as age,condition, sex and extent of disease, if any, and other variables whichcan be adjusted by one of ordinary skill in the art. The antigenicpolypeptide preparations of the invention can be administered as eithersingle or multiple dosages and can vary from about 50 mg to about 500 mgof the polypeptide antigen per dose, more preferably about 50 mg toabout 300 mg per dose, most preferably about 100 mg to about 200 mg perdose. The monoclonal antibodies of the invention can also be used, aloneor in combination with effector cells (Douillard, et al., Hybridoma, 5(Supp. 1: S139, 1986), for immunotherapy in an animal havingEBV-associated disease with epitopes reactive with the monoclonalantibodies of the invention.

When used for immunotherapy, the monoclonal antibodies of the inventionmay be unlabeled or labeled with a therapeutic agent. These agents canbe coupled either directly or indirectly to the monoclonal antibodies ofthe invention. One example of indirect coupling is by use of a spacermoiety. These spacer moieties, in turn, can be either insoluble orsoluble (Diener, et al., Science, 231:148, 1986) and can be selected toenable drug release from the monoclonal antibody molecule at the targetsite. Examples of therapeutic agents which can be coupled to themonoclonal antibodies of the invention for immunotherapy are drugs,radioisotopes, lectins, and toxins.

The drugs with which can be conjugated to the monoclonal antibodies ofthe invention include non-proteinaceous as well as proteinaceous drugs.The terms “non-proteinaceous drugs” encompasses compounds which areclassically referred to as drugs, for example, mitomycin C,daunorubicin, and vinblastine.

The proteinaceous drugs with which the monoclonal antibodies of theinvention can be labeled include immunomodulators and other biologicalresponse modifiers. The term “biological response modifiers” is meant toencompass substances which are involved in modifying the immuneresponse, for example, in such manner as to enhance the destruction ofan EBV-associated disease cell having an EBV antigen comprising apolypeptide of the invention for which the monoclonal antibodies of theinvention are specific. Examples of immune response modifiers includesuch compounds as lymphokines. Lymphokines include tumor necrosisfactor, interleukins 1, 2, and 3, lymphotoxin, macrophage activatingfactor, migration inhibition factor, colony stimulating factor, andinterferon. Interferons with which the monoclonal antibodies of theinvention can be labeled include alpha-interferon, beta-interferon, andgamma-interferon and their subtypes.

In using radioisotopically conjugated monoclonal antibodies of theinvention for immunotherapy certain isotypes may be more preferable thanothers depending on such factors as leukocyte distribution as well asisotype stability and emission. If desired, the tumor cell distributioncan be evaluated by the in vivo diagnostic techniques described above.Depending on the malignancy some emitters may be preferable to others.In general, alpha and beta particle-emitting radioisotopes are preferredin immunotherapy. For example, if an animal has solid tumor foci a highenergy beta emitter capable of penetrating several millimeters oftissue, such as ⁹⁰Y, may be preferable. On the other hand, if themalignancy consists of simple target cells, as in the case of leukemia,a short range, high energy alpha emitter, such as ²¹²Bi, may bepreferable. Examples of radioisotopes which can be bound to themonoclonal antibodies of the invention for therapeutic purposes are¹²⁵I, ¹³¹I, ⁹⁰Y, ⁶⁷Cu, ²¹²Bi, ²¹¹At, ²¹²Pb, ⁴⁷Sc, ¹⁰⁹Pd, and ¹⁸⁸Re.

Lectins are proteins, usually isolated from plant material, which bindto specific sugar moieties. Many lectins are also able to agglutinatecells and stimulate lymphocytes. However, ricin is a toxic lectin whichhas been used immunotherapeutically. This is preferably accomplished bybinding the alpha-peptide chain of ricin, which is responsible fortoxicity, to the antibody molecule to enable site specific delivery ofthe toxic effect.

Toxins are poisonous substances produced by plants, animals, ormicroorganisms that, in sufficient dose, are often lethal. Diphtheriatoxin is a substance produced by Corynebacterium diphtheria which can beused therapeutically. This toxin consists of an alpha and beta subunitwhich under proper conditions can be separated. The toxic A componentcan be bound to an antibody and used for site specific delivery to anEBV-antigen bearing cell for which the monoclonal antibodies of theinvention are specific. Other therapeutic agents which can be coupled tothe monoclonal antibodies of the invention are known, or can be easilyascertained, by those of ordinary skill in the art.

The labeled or unlabeled, monoclonal antibodies of the invention canalso be used in combination with therapeutic agents such as thosedescribed above. Especially preferred are therapeutic combinationscomprising the monoclonal antibody of the invention and immunomodulatorsand other biological response modifiers. Thus, for example, themonoclonal antibodies of the invention can be used in combination withalpha-interferon. This treatment modality enhances monoclonal antibodytargeting of EBV-containing cells by increasing the expression ofmonoclonal antibody reactive antigen (Greiner, et al., Science, 235:895,1987). Alternatively, the monoclonal antibody of the invention could beused, for example, in combination with gamma-interferon to therebyactivate and increase the expression of Fc receptors by effector cellswhich, in turn, results in an enhanced binding of the monoclonalantibody to the effector cell and killing of target tumor cells. Thoseof skill in the art will be able to select from the various biologicalresponse modifiers to create a desired effector function which enhancesthe efficacy of the monoclonal antibody of the invention.

When the monoclonal antibody of the invention is used in combinationwith various therapeutic agents, such as those described herein, theadministration of the monoclonal antibody and the therapeutic agentusually occurs substantially contemporaneously. The term “substantiallycontemporaneously” means that the monoclonal antibody and thetherapeutic agent are administered reasonably close together withrespect to time. Usually, it is preferred to administer the therapeuticagent before the monoclonal antibody. For example, the therapeutic agentcan be administered 1 to 6 days before the monoclonal antibody. Theadministration of the therapeutic agent can be daily, or at any otherinterval, depending upon such factors, for example, as the nature of thetumor, the condition of the patient and half-life of the agent.

Using the monoclonal antibodies of the invention, it is possible todesign therapies combining all of the characteristics described herein.For example, in a given situation it may be desirable to administer atherapeutic agent, or agents, prior to the administration of themonoclonal antibodies of the invention in combination with effectorcells and the same, or different, therapeutic agent or agents. Thus, itmay be desirable to treat patients with leukemia or lymphoma by firstadministering gamma-interferon and interleukin-2 daily for 3 to 5 days,and on day 5 administer the monoclonal antibody of the invention incombination with effector cells as well as gamma-interferon, andinterleukin-2.

It is also possible to utilize liposomes with the monoclonal antibodiesof the invention in their membrane to specifically deliver the liposometo the area of the EBV-associated disease cell. These liposomes can beproduced such that they contain, in addition to the monoclonal antibody,such immunotherapeutic agents as those described above which would thenbe released at the tumor site (Wolff, et al., Biochemical et BiophysicalActa, 802:259, 1984).

The dosage ranges for the administration of the monoclonal antibodies ofthe invention are those large enough to produce the desired effect inwhich the symptoms of the EBV-associated disease are ameliorated. Thedosage should not be so large as to cause adverse side effects, such asunwanted cross-reactions, anaphylactic reactions, and the like.Generally, the dosage will vary with the age, condition, sex and extentof the disease in the patient and can be determined by one of skill inthe art. The dosage can be adjusted by the individual physician in theevent of any complication. Dosage can vary from about 0.1 mg/kg to about2000 mg/kg, preferably about 0.1 mg/kg to about 500 mg/kg, in one ormore dose administrations daily, for one or several days. Generally,when the monoclonal antibodies of the invention are administeredconjugated with therapeutic agents lower dosages, comparable to thoseused for in vivo immunodiagnostic imaging, can be used.

The monoclonal antibodies of the invention can be administeredparenterally by injection or by gradual perfusion over time. Themonoclonal antibodies of the invention can be administeredintravenously, intraperitoneally, intra-muscularly, subcutaneously,intracavity, or transdermally, alone or in combination with effectorcells. Preparations for parenteral administration include sterileaqueous or non-aqueous solutions, suspensions, and emulsions.

Examples of non-aqueous solvents are propylene glycol, polyethyleneglycol, vegetable oils such as olive oil, and injectable organic esterssuch as ethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

The invention also relates to a method for preparing a medicament orpharmaceutical composition comprising a polypeptide, or a monoclonalantibody of the invention, the medicament being used for therapy ofEBV-associated disease.

Reactivity of a sample with EA-D, 50.10 peptides or anti-EA-D peptideantibodies is preferably associated with nasopharyngeal carcinoma andinfectious mononucleosis. Likewise, reactivity with EA-R, 17.1 peptidesor anti-EA-R peptide antibodies is preferably associated with lymphomas.These specific peptides and their corresponding monoclonal antibodiesare particularly useful for detecting the EA-D and EA-R transitionsassociated with a particular disease state.

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific examples which are provided for purposes of illustration only,and are not intended to limit the scope of the invention.

EXAMPLE 1 Synthesis of p17 and p50 Polypeptides

Potential T cell epitopes on the p17 protein were identified usingBerzofsky's algorithm (AMPHI Program) (1987). This algorithm postulatesthat T-cells preferentially interact with peptides that are amphipathicand which form an alpha helical configuration. Based on thesecharacteristics, candidate epitopes on the p17 and p50 proteins weremapped and were found scattered throughout the molecule. The epitopeswith the highest amphipathic scores were synthesized and employed in thestudies. Out of 8 predicted epitopes on p17, the 3 with the highestscores were synthesized as 15 amino acid residues based on thenucleotide sequences encoding for these putative epitopes (FIG. 1, Table1). Peptide synthesis was carried out using the solid phase method ofMerrifield (1963) on an Applied Biosystems ABI 430-A automated peptidesynthesizer using hydroxybenzotrizole hydrate/dicyclohexylcarbodiimideactivation as described (Curtiss, L. K., et al., J. Biol. Chem.,263:13779-13785, 1988). The resultant peptide resins were treated with10% anisole/hydrogen fluoride at −4° C. for 1 hour (Lenard, J., et al.,J. Am. Chem. Soc., 89:181-182, 1967). The peptide preparations (10 μgper run) were analyzed by HPLC using a VYDAC C¹⁸ column. The startingbuffer contained 0.1% trifluoroacetic acid in water (solvent A) and 0.1%trifluoroacetic acid in acetonitrile (solvent B). The run consisted of a20-70% gradient increase in solvent B over 20 minutes at 40° C. Theseparations were monitored at an absorbance of 214 nm. Preparativepurification of the peptides employed chromatography on a WATERS AUTO500 preparative HPLC (50×250-mm VYDAC C¹⁸ column, 15-20 μm) under thesame conditions as described for analytical chromatography. Amino acidcompositions of al peptides were determined after hydrolysis with aBeckman 6300 high performance analyzer with internal standards. Allpeptides were lyophilized and stored under vacuum. Synthesis of p50polypeptides was performed as described above for p17.

TABLE 1 AMINO ACID SEQUENCE OF SYNTHETIC PEPTIDESFROM 17 Kd EARLY ANTIGEN (EA-R) REGIONAND 50 Kd EARLY ANTIGEN (EA-D) REGION Location^(a) PeptideAmino Acid Sequences Genomic Peptide p17.1 ETFTETWNRFITHTE 54.562-54.60462-76 (SEQ. I.D. NO. 1) p17.2 GMLEASEGLDGWIHQ 54.766-54.808 130-144(SEQ. I.D. NO. 2) p17.3 HQQGGWSTLIEDNIP 54.805-54.847 143-157(SEQ. I.D. NO. 3) p50.10 KQKHPKKVKQAFNPL 81.063-81.108 —(SEQ. I.D. NO. 4) p17.1^(b) QNSETFTETWNRFITHTEHVD 54.553-54.613 59-79(SEQ. I.D. NO. 5) p50.10^(b) ARQKQKHPKKVKQAFNPLI 81.054-81.111 —(SEQ. I.D. NO. 6) ^(a)Location based on the predicted position ofnucleotides in the prototype EBV (B95.8) DNA sequence of Baer et al.,(Nature 310:207, 1984). ^(b)Extension of p17.1 and p50.10, respectively,by the addition of several amino and carboxy-terminal amino acids.

EXAMPLE 2 Proliferative Response of PBL to Synthetic Peptides

Cells. The P₃HR-1 cell line, established from an African Burkitt'slymphoma (ABL) biopsy, was the source of the native p17 component of theEA-R complex in these experiments (Himuna, Y., et al., J. Virol.,1:1045-1051, 1967). The cells were grown in the presence of RPMI 1640medium supplemented with 10% heat-inactivated (56° C., 30 minutes) fetalcalf serum (FCS), 2 mM L-glutamine and 50 μg per ml gentamycin at 37° C.The cells were passaged every 3-4 days by dilution with fresh medium toa cell concentration of 5×10⁵ cells per ml.

For antigen production, P₃HR-1 cells were activated with 20 ng per mlTPA (12-0-tetradecanoyl-phorbol-13 acetate) and 3 mM sodium butyrate for48 hours. This procedure generally results in the induction ofexpression of this antigen in more than 70% of the cells as determinedby immunofluorescence (Pearson et al., Virol., 160:151-161, 1987).

ELISA. The ELISA for measuring specific antibodies to p17, p50 or to thesynthetic peptides was performed as previously described in detail (Lukaet al., J. Immunol. Methods, 67:145-156, 1984). Aliquots of differentantigen concentrations were diluted in 0.5M Na₂CO₃ buffer, pH 9.5, addedto wells in polystyrene microtiter plates (Linbro) and the platesincubated overnight at 4° C. Following this incubation period, theplates were washed 5× with Tris-HCl, pH 7.4, containing 0.05% Tween 20,50 mM NaCl and 100 mg per liter albumin (Sigma) and dried for 20 minutesat room temperature. The plates were screened with different anti-EA-Ror EA-D antibody positive human sera and the monoclonal antibody to p17or p50 to identify the optimal concentration of antigen to be used inthe serological studies. Alkaline-phosphatase-labelled goat anti-humanIgG (Sigma) or goat anti-mouse IgG (Sigma) was used as the indicatorsystem.

For the testing of human sera for antibodies to native p17, p50 or tothe synthetic peptides, sera were diluted 1:10 in ELISA buffer, added in0.1 ml volumes to wells coated with optimal concentrations of antigenand the plates were incubated for 60 minutes at room temperature. After4 washes with ELISA buffer, 100 μl of alkaline phosphatase-labelled goatanti-human IgG in ELISA buffer were added to each well and the plateswere incubated at room temperature for 1 hour. Following four morewashes in buffer, the enzyme reaction was performed by dissolving 1 mgper ml of Sigma alkaline phosphatase substrate in 1M diethanolaminebuffer, pH 10.4, containing 1 mM MgCl₂ and 0.1 mM ZnCl₂, and 100 μl ofthe mixture was then added to each well. The reaction was allowed toproceed for 30 minutes at 37° C. and then the plates were screeneddirectly with a microplate reader, TITERTEK MULTISKAN MC (Flow), at 420nm. Readings above 0.1 which was 2× the background noted withantibody-negative sera were considered positive reactions.

Purification of native p17. Cells expressing p17 were washed twice inphosphate-buffered saline (PBS), resuspended in an extraction buffercontaining 0.5% NP-40 and 0.5% sodium deoxycholate in 0.02 M Trishydrochloride (pH 7.4), 0.15 M NaCl, 1 mM B-mercaptoethanol and 10 mMphenylmethyl-sulfonylfluoride (PMSF) and sonicated for 5 cycles of 20second each. The extracts were then clarified by centrifugation at40,000×g for 60 minutes at 4° C. in a Beckman JA-20 rotor and theresultant cell-free supernatant passed over an affinity column preparedwith a monoclonal antibody to p17 (Pearson et al., Cancer, 51:260-268,1983). The column was washed once with 5 volumes of extraction bufferand then once with buffer without detergents before elution of the boundp17 with 3M MgCl₂ buffered with 20 mM Tris-HCl (ph 7.4). The eluateswere tested for specificity by Western blotting and ELISA and titratedfor specific antigen activity by ELISA (Luka et al., J. Immunol.Methods, 67:145-156, 1984). The protein concentrations were determinedusing the Bio-Rad assay. The p17-containing eluates were then dialyzedagainst 100 volumes of 10 mM Tris-HCl (pH 7.4), 150 mM NaCl followed bydialysis against RPMI 1640 medium containing 10% human A EBVantibody-negative serum. The antigen was aliquoted and stored at −70° C.until used in the different immunological assays. (Purification of p50was performed as described above for p17.)

Proliferation assays. T-cell proliferation assays using the native p17or p50 polypeptide or the synthetic peptides were performed aspreviously described in detail (Pothen et al., Int. J. Cancer,49:656-660, 1991). Briefly, peripheral blood lymphocytes (PBLO fromsero-positive donors were isolated on FICOLL-HYPAQUE gradients, washedand resuspended in RPMI 1640 media containing 10% heat-inactivated humanA⁺ serum from a sero-negative donor. Cells (1×10⁵ per 0.1 ml) were thenadded to each well in 96-well, round-bottomed tissue culture plates(Costar, Cambridge, Mass.). The different antigen preparations wereadded to triplicate wells in 0.1 ml volumes and the plates wereincubated at 37° C. for 5 days. ³H-thymidine (5 Ci per mM) was added ata concentration of 1 μCi per well over the last 4 hours of culture. Thecells were then harvested with a multichannel sample harvester and³H-thymidine incorporation was determined using a Beckman Model LS 3801liquid scintillation counter. Stimulation indices for the test antigenswere calculated by dividing the average counts per minute (CPMO for thetest antigens by the average CPM of the medium control wells.

PBL from asymptomatic EBV-infected individuals were tested for responseto any of the three p17 synthetic peptides (Table 1) by a proliferationassay using lymphocytes previously shown to proliferate in the presenceof native p17 (Pothen et al., Int. J. Cancer, 49:656-660, 1991).Different concentrations of the synthetic peptides were incubated withthe PBL for 5 days and then proliferation was assayed by theincorporation of ³H-thymidine. Results from this initial experimentusing PBL from an anti-VCA-positive, anti-EA-positive donor are shown inTable 2. The native p17 purified from activated P₃HR-1 cells gave a S.I.of 15.8 in this experiment. At concentrations of 50 and 12.5 μg per ml,the p17.1 peptide also induced significant proliferation responses(S.I.'s of 10.8 and 4.7 respectively). Neither of the other syntheticpeptides induced a proliferation response at the concentrations tested.PBL from an EBV antibody-negative individual also did not respond to anyof the synthetic peptides.

PBL from 3 anti-VCA-positive anti-EA-antibody-negative individuals werealso examined in the proliferation assay with the three syntheticpeptides. These results are shown in FIGS. 2A, 2B, and 2C. Again, all 3PBL preparations responded to the highest concentrations of p17.1 withS.I.'s ranging from 3.5-11. Two of the preparations (FIG. 2A,C) alsoproliferated in the presence of lower concentrations of antigen withS.I.'s of 3 and 5 respectively. None of these PBL preparationsproliferated in the presence of p17.2 and p17.3. These experimentsestablished that T-lymphocytes from EBV-infected individuals,irrespective of the presence of antibody to EA, recognized a dominantepitope on p17.

TABLE 2 PROLIFERATION RESPONSE OF PERIPHERAL BLOOD LYMPHOCYTES FROM ANEBV-INFECTED DONOR TO P17 SYNTHETIC PEPTIDES ³H-thymidine ConcentrationIncorporation Antigen¹ (μg/ml) (cpm + S.D.) S.I. — — 147 ± 78 — PHA 10391567 ± 55800 — Native p17² 35 2327 ± 73  15.8 p17.1 50 1584 ± 37  10.812.5 689 ± 88 4.7 3.12 263 ± 57 1.8 p17.2 50  277 ± 175 1.9 12.5 278 ±58 1.9 3.12 157 ± 23 1.1 p17.3 50 190 ± 47 1.3 12.5 132 ± 3  0 3.12 194± 65 1.3 ¹Antigen was incubated for 5 days with 1 × 10⁵ lymphocytes.Counts per minute (cpm) ± standard deviation (S.D.) determined fromtriplicate cultures. ²Purified from P₃HR-1 cells by immunoaffinitychromatography.

EXAMPLE 3 Fractionation of PBL into CD4⁺ and CDB⁺ Subpopulation

PBL enriched for T-cell subpopulations were isolated by depleting aparticular population by antibody/complement-mediated cytotoxicity. CD4⁺cells were prepared by lysing CD8⁺ cells with anti-CD8 antibody andrabbit complement whereas the CD8⁺ T cell subpopulation was enriched bylysing CD4⁺ cells with an anti-CD4 antibody and rabbit complement.

PBL were resuspended in RPMI-1640 medium containing 2 mM L-glutamine, 25mM HEPES, and 10 μg per ml gentamicin (HEPES media) at a concentrationof 20×10⁶ cells per ml. T cell specific MAb, OKT4 or OKT8 (OrthoDiagnostics Inc.), was added to the cells at the optimal concentration(previously determined by titration to give maximal lysis) and incubatedon ice for 30 minutes. The cells were then washed with fresh HEPES mediaand resuspended at 10×10⁶ cells per ml in baby rabbit complement(Pel-Freez Clinical Systems) diluted with the HEPES media to theappropriate concentration (previously assayed to give maximalantibody-specific cytotoxicity). The cells were incubated for 45 minutesin a 37° C. water bath with gentle mixing every 15 minutes. Afterincubation, the cells were washed thoroughly with the HEPES media and analiquot (1-2×10⁶) of cells was removed and stained for flow cytometricanalysis which was performed as described below. PBL that were notincubated with either antibody or complement were also processed inparallel for flow cytometry in order to ascertain the originalpercentage of CD4⁺ and CD8⁺ cells in each donor. The populations thatyielded >95% purity and viability were used in proliferation assays.

Proliferation assays using the CD4⁺ or CD8⁺-enriched PBL from theseropositive donors were conducted with varying concentrations of thesynthetic peptides as described above. However, in these experimentsadditional irradiated autologous PBL were used as antigen presentingcells (APC) at a ratio of 5×10⁵ APC to 1×10⁵ CD4⁺ or CD8⁺ cells.

FACS Analysis. The cells removed after incubation above (1-2×10⁶), werestained for 45 minutes at 4° C. with 20 μl of the SIMULTEST reagent(Becton-Dickinson) containing fluorescein isothiocyanate conjugatedantileu 3a (CD4 marker) and phycoerythrin-conjugated anti-leu 2a (CD8marker). Cells were washed thoroughly, resuspended in RPMI-1640 with 5%FCS and 0.02% sodium azide and analyzed with the cell sorter (FACSTARPlus, Becton-Dickinson).

To determine whether both CD4⁺ and CD8⁺ T-cell subpopulations wereresponding to p17.1, lymphocytes from two donors were separated intothese two subpopulations which were then employed in the proliferationassay. Results are presented in FIGS. 3A and 3B. The PBL from bothdonors proliferated in the presence of p17.1 at the highestconcentrations tested in this experiment (100 μg per ml for donor A and50 μg per ml for donor B). The CD4⁺ subpopulation from donor A alsoresponded vigorously to different concentrations of p17.1 with S.I.'s ashigh as 5.3. The CD8⁺ subpopulation from this donor was unresponsive tothis synthetic peptide. This pattern of response was also observed withfractionated CD4⁺ and CD8⁺ T-cells from another seropositive donor. Incontrast, both the CD4⁺ and CD8⁺ T-cell subpopulations from donor Bresponded to p17.1 with the CD8⁺ subpopulations giving a S.I. of greaterthan 3 at the highest antigen concentration tested (50 μg per ml). Theseresults therefore indicated that both CD4⁺ and CD8⁺ T-cells recognizedthis p17 epitope.

EXAMPLE 4 Serological Response to p17 Synthetic Epitopes

Studies were designed to determine the serological reactivity with thep17 synthetic epitopes. For this purpose, 87 anti-EA antibody-positivesera (titer>160) were tested in the ELISA against optimal pre-determinedconcentrations (2 μg per well) of the p17 synthetic peptides. The serawere obtained from 28 patients with African Burkift's lymphoma (ABL)collected during the Ghanian Burkitt Tumor Project, and from 28 NorthAmerican patients with intermediate large cell, or high gradenon-Hodgkin's lymphoma (NANHL). The donors included both HIV-positiveand HIV-negative individuals. In addition, sera from 31 North Americannasopharyngeal carcinoma (NANPC) patients were examined in this study(Pearson, G. R., et al., Cancer, 51:260-268, 1983). To validate the EAspecificity of the serological reactions, the results with these serawere compared with results with 23 VCA and EA antibody-negative sera and30 VCA-antibody-positive, EA-antibody negative sera. Results are shownin FIG. 4. In contrast to the T-cell proliferation results, all threesynthetic peptides reacted with the EA antibody-positive sera to varyingdegrees with p17.1 again being the dominant epitope. Approximately 60%of the anti-EA-positive sera reacted with p17.1, 48% with p17.2 and 23%with p17.3. The anti-EA specificity of these serological results wasassured by the lack of reactivity of any of the 53 anti-EAantibody-negative sera with any of the three synthetic peptides.Therefore, these results identified three B-cell epitopes on p17.

To determine why some anti-EA-antibody sera reacted with these syntheticpeptides while others did not, the data were analyzed according to theserum donor. These results are presented in FIG. 5. A strikingspecificity was noted with patients with lymphoproliferative diseaseswho normally have anti-EA-R antibodies in their sera as opposed to theanti-EA-D-positive NPC. Seventy-one percent of the sera from ABLpatients and 93% of the sera from patients with NANHL reacted with p17.1as opposed to 23% of the sera from North American patients with NPC. Asimilar specificity was noted with p17.2 and p17.3. These resultsdemonstrated the potential value of one or more of these syntheticpolypeptides for measuring anti-EA-R antibodies in the sera of patientswith lymphoproliferative diseases and are summarized in Table 3.

TABLE 3 SUMMARY OF SERUM REACTIVITY WITH 17 KD EA-R SYNTHETIC PEPTIDESNo. Positive/No. Tested (%) Serum Donors¹ p17.1 p17.2 p17.3 Normal (VCA− EA-) 0/23 (0) 0/23 (0) 0/23 (0) Normal (VCA + EA-) 0/30 (0) 0/30 (0)0/30 (0) Lymphoma (total) 46/56 (82) 36/56 (64) 16/56 (29) a) Burkitt'slymphoma 20/28 (71) 13/28 (46)  9/28 (32) (African) b) Non-Hodgkin'slymphoma 26/28 (93) 23/28 (82)  7/28 (25) (North American)Nasopharyngeal carcinoma  7/31 (23)  6/31 (19)  4/31 (13) (NorthAmerican) ¹All lymphoma and nasopharyngeal carcinoma serum donors hadantibody titers to EA which were >160 as determined byimmunofluorescence. These sera also were positive for antibodies to thenative 17 Kd EA-R protein purified from P₃HR-1 cells as determined byELISA.

EXAMPLE 5 p50.10 from the EA-D Region

The 50 Kd protein found in the EA-D region of EBV contains a dominantepitope called 50.10. This epitope is located on the EBV genome at81.063 to 81.108 based on the prototype EBV (B95.8) DNA sequence ofBaer, et al., supra. (See Table 1, Example 1.) Synthetic peptides wereprepared for 50.10 according to the method described in Example 1. Theamino acid sequence of the synthetic peptide for this epitope is

KQKHPKKVKQAFNPL. (SEQ. I.D. NO. 4)¶  A       B

The region of the epitope from 81.036-81.078, which includes portion A,underlined above, shows only weak reactivity with infectiousmononucleosis (IM) sera, while the region from 81.081-81.120, whichcontains portion B, shows no reactivity with 1M sera. When the entire50.10 peptide was tested with 1M sera, 24/32 or 75% of the seracontained EA-D reactive antibody (IgG) (Table 4). In comparison, only12/32, or 38% of IM sera tested showed EA-R reactive antibody.

TABLE 4 SCORING EBV IM WITH EA PEPTIDES 17.1 AND 50.1 EA-R PEPTIDE 17.1EA-D PEPTIDE 50.10 SAMPLE NO. O.D. SCORE O.D. SCORE 2241 0.282 − 0.673 +11984 0.542 + 0.722 + 12005 0.981 + 0.441 − 12288 0.700 + 0.610 + 124070.787 + 0.751 + 12409 0.592 + 0.609 + 12418 0.463 − 0.382 − 124980.637 + 0.342 − 12502 0.924 + 0.270 − 12587 0.571 + 0.890 + 12600 0.260− 0.323 − 12611 0.259 − 0.436 − 12801 0.601 + 0.605 + 12828 0.375 −0.895 + 12829 0.257 − 0.652 + 12836 0.692 + 0.722 + 12837 0.523 +1.491 + 12840 0.488 − 0.723 + 12888 0.423 − 0.847 + 12889 0.574 +1.184 + 12892 0.430 − 0.634 + 12893 0.592 + 0.626 + 12899 0.825 +0.895 + 12900 0.264 − 0.498 − 13075 0.154 − 0.252 − 13419 0.655 +0.678 + 13637 0.719 + 0.886 + 13769 0.757 + 1.307 + 13929 0.332 −1.031 + 14043 0.095 − 0.631 + 14128 0.434 − 0.693 + 14131 0.182 −0.530 + % Positive 12/32 = 38% 24/32 = 75% Values >0.5 were consideredpositive (O.D. readings at 420 nm).

Serological reactivity of EA peptides p17.1 and p50.1 which containedadditional amino acids on their amino and carboxy termini was tested onEBV IM acute sera which was EA positive. The ELISA results shown inTable 5 indicate that the additional amino acids improved reactivity forboth EA peptides.

TABLE 5 COMPARISON OF EA PEPTIDES 17.1 AND 50.1 WITH EXTENSIONS IN EAPOSITIVE EBV IM ACUTES EA-R EA-D Sample No. 17.1 17.1e 50.1 50.1e 22410.282 1.277 0.673 >2 12498 0.637 0.502 0.342 0.946 12836 0.692 1.2570.722 1.718 e = extension as described in Table 1.

EXAMPLE 6 Serological Results with p50.10

Serological reactivity of different sera (VCA−EA−, VCA+EA+, VCA+EA−)with the p50.10 synthetic peptide was performed as described in Example1, according to Pothen, et al., supra. The anti-EA specificity of theserological result was confirmed by lack of reactivity of p50.10 peptidewith anti-EA antibody negative sera. Therefore, these results show aB-cell epitope on p50.

Immunofluorescence assays (IFA) were performed as described by Henle, etal. (Int. J. Cancer, 8:272-282, 1971). Western blot analysis was done bymethods commonly used in the art (See for example, Coligan, et al.,Current Protocols in Immunology, Wiley Interscience, 1991, Unit 12).Samples of EBV-infected cell lysates (P₃HR-1) were tested with humanwhole antiserum from positive humans (Pearson, et al., Virology,160:151-161, 1987). A comparison was made between EA serology by IFA,using the EA-D 50.1 peptide antisera, and ELISA serology using 50.1peptide (see Example 2). Samples tested were IM positive and EA-positiveby Western blot analysis (50-55K positive reactivity), however, as seenin Table 6, the samples were IFA-EA negative, even at a dilution of1:10.

TABLE 6 COMPARISON OF EPSTEIN-BARR VIRUS EARLY ANTIGEN (EA) SEROLOGY BYIMMUNOFLUROESCENCE TO THE EA-D 50.1 SYNTHETIC PEPTIDE ELISA¹ SampleNumber IFA EA Test 50.1 ELISA² 12502  <10¹ 0.409 12840 <10 0.257 13769<10 1.034 ¹Performed on acute EBV infectious mononucleosis samples(diluted 1:10) that were EA-D Western blot positive ²OD reading greaterthan 0.2 were considered positive.

The serological reactivity of sera from 1) anti-EA-negative,VCA-negative (viral capsid antigen); 2) anti-EA-R positive lymphomas;and 3) anti-EA-D positive, nasopharyngeal carcinomas were studied byELISA. As seen in Table 7, there is a high correlation between p17.1reactivity and lymphomas and p50.10 reactivity and nasopharyngealcarcinoma.

TABLE 7 COMPARATIVE REACTIVITY OF ANTI-EA-R AND ANTI-EA-D SERA AGAINSTEA-R (P17.1) AND EA-D (P50.10) SYNTHETIC PEPTIDES ELISA (420 nm)¹ Serumdonor p17.1 p50.10 Anti-EA-negative 0.071 0.089 (VCA-negative) 0.0270.098 0.024 0.154 0.041 0.086 Anti-EA-R-positive 0.682 0.079 (Lymphomas)1.354 0.078 1.793 0.098 1.218 0.817 1.056 0.318 1.104 0.095 Anti-EA-Dpositive 0.019 1.482 (Nasopharyngeal 0.014 1.573 carcinoma) 0.041 >2.00.025 1.923 0.265 0.944 0.029 >2.0 ¹OD readings at 420 nm; valuesgreater than 0.2 were considered significant.

EXAMPLE 7 Detection of Early Antigen D-R Transition

The early antigen D-R transition was followed in a patient during acourse of acute infectious mononucleosis. Antibodies to peptides 50.10(EA-D) and 17.1 (EA-R) were detected by ELISAs on specimens collectedover a six month duration. The data in Table 8 show that the antibodiesto peptide 50.10 peaked at 6-27-88, while the antibodies to peptide 17.1peaked at 7-18-88. Therefore, the peptides of the invention are usefulfor monitoring the course of EBV-associated disease in a patient.

TABLE 8 IgG ELISA: SEQUENTIAL SERA IgG¹ DATE SERUM NO. p17.1 p.50.10 May26, 1988 9817 0.158 0.104 May 31, 1988 9820 0.106 0.094 Jun. 07, 19889829 0.135 0.120 Jun. 20, 1988 9941 0.169 0.147 Jun. 27, 1988 9983 0.6450.817 Jul. 18, 1988 10064 0.842 0.821 Aug. 29, 1988 10237 0.260 0.561Sep. 20, 1988 10271 0.231 0.100 Oct. 21, 1988 10488 0.217 0.095 Nov. 15,1988 10546 0.193 0.140 ¹O.D. reading; sera tested at 1.20 dilution; (+)>0.25.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. Variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.

SUMMARY OF SEQUENCES

Sequence ID No. 1 is the amino acid sequence for the peptide, p17.1,from the 17 Kd early antigen (EA-R) region of EBV (Page 7, line 3, Page29, line 11 in Table 1; Page 39, line 8; and Page 41, line 3).

Sequence ID No. 2 is the amino acid sequence for the peptide, p17.2,from the 17 Kd early antigen (EA-R) region of EBV (Page 7, line 3; Page29, line 12 in Table 1; and Page 41, line 4).

Sequence ID No. 3 is the amino acid sequence for the peptide, p17.3,from the 17 Kd early antigen (EA-R) region of EBV (Page 7, line 3; Page29, line 13 in Table 1; and Page 41, line 5).

Sequence ID No. 4 is the amino acid sequence for the peptide, p50.10,from the 50 Kd early antigen (EA-D) region of EBV (Page 7, line 4; Page29, line 14 in Table 1; and Page 41, line 6).

1. A monoclonal antibody to a polypeptide consisting of an amino acidsequence having the formula:(φ)_(n) wherein n is 1 to about 1000 and φ is 19 amino acids or less andhas the formula: (α ETFTETWNRFITHTE β)_(n) (SEQ. ID NO: 7) (αGMLEASEGLDGWIHQ β)_(n) (SEQ. ID NO: 8) (α HQQGGWSTLIEDNIP β)_(n)(SEQ. ID NO: 9) (α KQKHPKKVKQAFNPL β)_(n) (SEQ. ID NO: 10)

wherein α and β are independently from 0 to about 2 naturally occurringamino acids wherein the polypeptide is capable of binding antibody in aspecimen from an individual with Epstein-Barr virus (EBV)-associateddisease.
 2. A hybridoma cell line capable of producing the monoclonalantibody of claim
 1. 3. A pharmaceutical composition comprising at leastone dose of a therapeutically effective amount of a monoclonal antibodyof claim 1 in a pharmacological carrier.
 4. The pharmaceuticalcomposition of claim 3 wherein the monoclonal antibody is human.
 5. Akit useful for detection of an amino acid sequence comprising apolypeptide consisting of an amino acid sequence with the formula:(φ)_(n) wherein n is 1 to about 1000 and φ is 19 amino acids or less andhas the formula: (α ETFTETWNRFITHTE β)_(n) (SEQ. ID NO: 7) (αGMLEASEGLDGWIHQ β)_(n) (SEQ. ID NO: 8) (α HQQGGWSTLIEDNIP β)_(n)(SEQ. ID NO: 9) (α KQKHPKKVKQAFNPL β)_(n) (SEQ. ID NO: 10)

wherein α and β are independently from 0 to about 2 naturally occurringamino acids, wherein the polypeptide is capable of binding antibody in aspecimen from an individual with Epstein-Barr virus (EBV)-associateddisease in a specimen suspected of containing such sequence, the kitcomprising carrier means being compartmentalized to receive in closeconfinement therein one or more containers comprising a containercontaining a monoclonal antibody to said polypeptide.